Nardilysin-regulated scission mechanism activates polo-like kinase 3 to suppress the development of pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) develops through step-wise genetic and molecular alterations including Kras mutation and inactivation of various apoptotic pathways. Here, we find that development of apoptotic resistance and metastasis of KrasG12D-driven PDAC in mice is accelerated by deleting Plk3, explaining the often-reduced Plk3 expression in human PDAC. Importantly, a 41-kDa Plk3 (p41Plk3) that contains the entire kinase domain at the N-terminus (1-353 aa) is activated by scission of the precursor p72Plk3 at Arg354 by metalloendopeptidase nardilysin (NRDC), and the resulting p32Plk3 C-terminal Polo-box domain (PBD) is removed by proteasome degradation, preventing the inhibition of p41Plk3 by PBD. We find that p41Plk3 is the activated form of Plk3 that regulates a feed-forward mechanism to promote apoptosis and suppress PDAC and metastasis. p41Plk3 phosphorylates c-Fos on Thr164, which in turn induces expression of Plk3 and pro-apoptotic genes. These findings uncover an NRDC-regulated post-translational mechanism that activates Plk3, establishing a prototypic regulation by scission mechanism.

1.The focus is on pancreatic cancer, but many results were obtained from 293T human embryonic kidney cell line (Figure 3, 4, 5, 6 and Supplementary Figure 2, 4) and human colorectal carcinoma cell line HCT116 (Supplementary Figure 2, 4).1b-1d: what are the levels of p41Plk3 in the metastatic cell lines?Supplementary figure 1c: whether -actin is analyzed on the same membrane?What are the levels for p41Plk3? 3.In figure 1b: among the cancer cell lines, 102, 108, and 124 also express a similar level of p72Plk3 as normal HPDE, while the expression of p72Plk3 appears to be relatively low in metastatic cell lines.So, whether loss of Plk3 is involved in metastasis specifically?4. What is the status of p41Plk3 in the KPC mice in Figure 1d? Figure 1h: is there an IHC staining done to show the expression of p41Plk3 in p48-cre; KrasLSL-G12DPlk3+/+. 5.In figure 1, most of the pancreatic cell lines still express a detectable p72Plk3 level.How do you justify this?Whether it determines the possibility of Plk3 independent tumor mechanism in PDAC or if there is any possible mechanism involved in inhibiting the function of p72Plk3.What's the expression pattern of NRDC in pancreatic cancer?6.In figure 2b -cleaved PARP induction is visibly seen after 2 hrs in suspension culture.Whereas figure 2i, 2j, 2k -HPDE/HEK293T/MEF cell lines reveal the cleavage to 41kd from 72 takes at least 3 hrs.Whether the induction of cleaved PARP is independent of 41kd? 7. The mouse developed a tumor in p48-cre; KrasLSL-G12DPlk3+/+ group, did it show any correlation with the genes mentioned in supplementary Table 1? Did authors analyze those samples because they should match with the genes mentioned in the surviving colonies that are resistant to Plk3 overexpression?8. What causes the Plk3 downregulation?Is there any known mutation?And what percentage of PDACs in which Plk3 is downregulated?If Plk1 seems to be an oncogene, is there an interplay between the two family members?9. Co-IP of NRDC indicated that LY294002 increased the binding of NRDC to centaurin-1 in the cytoplasm and decreased its binding to p72Plk3 (Fig. 4i).But it was shown only in 293T, which is a human embryonic kidney cell line, and not in any other PDAC cell line.Though the experiments with LY294002 were performed in many different cell lines, the Western for centaurin-1 was shown only in 293T cells.10. Figure 5i: What is the protein half-life of plk3NT/plk3CT in the metastasis cell lines?Whether different antibodies are used for detecting NT-1-354 and CT-356-646? 11.Supplementary Figure 7h -please mark the different cell cycle phases and include population distributions.

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12. Authors have found that p41Plk3 expression is significantly increased in the PTEN-KO compared to PTEN-WT MEF cells.It is also suggested that PI3K is required to cleave p72Plk3 into its active form p41Plk3 (apoptosis inducer).
-If p72Plk3 is a tumor suppressor and its active form p41Plk3 is anoikis or apoptosis inducer, then the concern is about PI3K because PI3K oncogenic signaling is known to be upregulated in PDAC.PTEN which is a known tumor suppressor KO increased p41Plk3 expression.Please justify how to correlate these events.
13. Plk1 cleavage is markedly increased in PTEN-KO but not in PTEN-WT MEFs.If Plk1 is a tumor promotor.It's been known that PTEN is a negative regulator of PI3K and PI3K is a known downstream effector of K-Ras, which is a driver of PDAC.Then how the KO of PTEN increases the cleavage of Plk3 into the active pro-apoptotic p41Plk3 KDa form?And vice versa how the inhibition of PI3K by LY294002 prevents the cleavage of p41plk3, which can induce apoptosis?14.Then if Plk1 is a tumor promotor and Plk3 is a tumor suppressor?What is the NRDC binding affinity to each.
Reviewer #3 -Plks, mass-spec -(Remarks to the Author): The manuscript was from the labs of Drs.Paul Chiao, Peter Stambrook and El Mustapha Bahassi, important contributors of the polo kinase field for over many years.The authors clearly deserved to be congratulated as this is another nice and important piece of contribution to the field.
The authors aimed to dissect the regulation of Plk3 in pancreatic cancer in great detail.Indeed, the regulation of Plk3 remained in the dark since its discovery and complete cloning in 2000 (Oncogene.2000 Oct 5;19(42):4832-9.doi: 10.1038/sj.onc.1203845).At first the authors analyzed the expression pattern of Plk3 in clinical samples and in the transgenic mouse models P48-Cre; KrasLSL-G12D; PLK3+/+ vs. Cre; KrasLSL-G12D; PLK3-/-.Upon overexpression of 72-kDa Plk3 associated with the loss of anchorage-dependent growth (high percentage of floating cells) a subfragment of Plk3 (p41Plk3) encompassing the kinase domain became visible.Plk3-induced anoikis correlated with increasing levels of p41Plk3.Most interestingly, the authors could identify via functional screening of an ORFs library the metalloendopeptidase nardilysin (NRDC) that is responsible for the cleavage of Plk3 into two subfragments.It is also of broad relevance that this mechanism plays a similar role for Plk1 cleavage.Furthermore, the regulation of NRDC by PI3-kinase was investigated in detail.C-Fos could be identified as novel substrate for Plk3 in its p41Plk3 form.
In summary, the data are of very high relevance for the development of pancreatic cancer and for the entire field of polo kinases.In addition, for the use of small molecule Plk inhibitors in clinical trials in relation to their specificity the new data by Fu et al. need to be considered.
A few aspects should be addressed before publication 1.Is there a correlation between mRNA levels of Plk3 and the corresponding protein in human pancreatic cancer (Fig. 1, Suppl.Fig. 1)?The authors should clearly state in which type of cancer cells (primary tissue, PDX cells etc.) this correlation was seen and what technique and which antibody was used.This aspect is important because there is a long-lasting debate about the quality of Plk3 antibodies.
2. Indeed, a role for Plk3 in cell cycle regulation has been discussed multiple times but not for the G2/M transition or the duration of mitosis.Thus, it is surprising that an overexpression of p41Plk3 induces a high percentage of cells (over 80%) that are stuck in mitosis.I think it would be beyond the scope of the manuscript to investigate the mechanism that is responsible for this mitotic arrest, but at least potential mechanisms should be discussed.
3. The major topic of this manuscript is the regulation of cell death by Plk3.The exact cell death pathways and mechanisms induced by Plk3 are still a matter for future research.In a previous study (Cell Res.2016 Aug;26(8):914-34.doi: 10.1038/cr.2016.78) the role of Plk3 for the regulation of the extrinsic cell death was explored.Would it be possible that p41Plk3 phosphorylates caspase-8 or other cell death transducing proteins in pancreatic cancer cells?Has this been explored?

Point-by-point Response
Dear Reviewers, Your review of our manuscript offered valuable comments and critiques.We thank you for your thoughtful suggestions and constructive criticisms.We did the experiments to address the critiques and revised the manuscript according your comments and suggestions.We presented the detailed discussions of revised experimental designs, results, and answers to all your questions.The key advances in the revised manuscript are briefly summarized as follows: • The in cis and in trans regulatory mechanisms for Plk1 and Plk3 activation revealed that both p41Plk3 and p38Plk1 are generated from the scission of catalytic inactive p72Plk3 and p68Plk1 kinases, respectively.
• Free from C-terminal Plk3 inhibitory effect, p41Plk3 elevates its kinase activity, phosphorylates c-Fos at Thr164, which in turn, increases the transcription of Plk3 and the pro-apoptotic genes and orchestrating cFos-Plk3 feed forward pathway, for regulating G2/M transition in cell cycle, inducing anoikis, and suppressing tumorigenesis and metastasis.
• As kinase domain is in a close proximity to PBD, activated p41Plk3 can be negatively regulated by the stabilized NRDC-cleaved Plk3 C-terminus in trans possibly through protein folding.
• Our findings clearly demonstrate that the PI3K-mediated activation of both Plk3 and Plk1 by NRDC proteolytic cleavage generates p41Plk3, which has a pro-apoptotic activity, and p38Plk1, which has prosurvival function, to maintain balance between the opposing effects of Plk3 and Plk1 kinases.
• Our results also revealed that p48-cre;Kras LSLG12D ;Plk3 -/-mice and "Dox/on" p72Plk3 mice developed metastatic PDAC, whereas ip72Plk3/NRDC or p41Plk3 expression impaired tumor growth, indicating that endopeptidase activity of NRDC is required for Plk3 activation and suppression of PDAC.
• Particularly striking is that cleavage by NRDC may play a key regulatory role in many signaling pathways.Analysis of 20,417 protein sequences in the UniProt SwissProt subset, we find that 5,833 proteins have no -X-Arginine-Lysine-NRDC cleavage sites (28.5%), 10,947 of them have 1-3 NRDC sites (53.6%), and 3637 (17.8%) of them have greater than 3 NRDC sites, suggesting that the -RKdibasic motif does not occur randomly (p<0.0001).
• There is a great potential that NRDC serves as an on/off signaling switch by cleaving its substrates, which contain structurally flexible binding motif and accessible cleavage sites close by, to activate or inactivate a signaling molecule.This discovery has potential far-reaching implications as a novel and important mechanism that orchestrates various biological signaling pathways through NRDC proteolytic activity.The regulatory potential of NRDC and its activation/inactivation of substrates by cleavage will be the focus of further exploration.
Thank you very much.
"In this manuscript, Fu et al. describe a role for decreased Plk3 expression in the formation and progression of PDAC as well as elucidate the mechanism by which Plk3 is cleaved and activated.The authors find that Plk3 expression is decreased in human PDAC compared with normal pancreatic tissues, and that genetic knockout of PDAC increases PDAC development in mice.Furthermore, Plk3 is cleaved in its DR1 site by nardilysin (NRDC) activity, which is subsequently results in an elevation in Plk3 kinase activity.Specifically, a 41 kDa form of Plk3 that contains the kinase active site in its n-terminal domain, known as p41Plk3, is sufficient to increase apoptotic death by phosphorylating c-Fos on T164 to direct it to the nucleus where it increases transcription for pro-apoptotic genes.Overexpression of this p41Plk3 subunit is also sufficient to decrease PDAC tumor burden and invasion in mice.
While the study is of interest, there are multiple concerns, both major and minor, that should be addressed prior to publication".
Major concerns 1. "In general, some conclusions made by the authors are overstated or otherwise not well supported by the data.For example, the figure caption of Fig. 2 states that "proteolytic processing of p72Plk3 to p41Plk3 is essential for induction of anoikis".However, the data as presented are merely correlative and indicate that p41Plk3 levels are associated with anoikis induction.Causality is never demonstrated." We thank the reviewer for raising these questions.In response to causality question, we have shown that the expression of Plk3 in HEK293T cells led to cell round-up, detaching from plate, floating and apoptosis (anoikis) (Supplementary Fig. 2a and 2b).The floating cells exhibited higher level of cleaved PARP than attached cells.While the vector-transfected 293 cells grown under attached and suspension condition do not exhibit cell death (Supplementary Fig. 2a and 2b).Further investigation of the mechanism through which Plk3 induces apoptosis led to the discovery of p41Plk3 with the level of p41Plk3 being higher in floating cells than in attached cells.(Supplementary Fig. 2b and 2i).These data suggest that the occurrence of p41Plk3 causes cell detachment-induced apoptosis, anoikis.
In the revised manuscript, to validate the essential role of p41Plk3 in inducing anoikis, we determined the effect of Plk3 deletion on PDAC cell anoikis (Fig. 2l, m).The result demonstrated that knockout of Plk3 expression in several PDAC cells led to decreased expression of p41Plk3 and subsequent reduced level of cleaved PARP and apoptosis-inducing activity (Fig. 2l, m).Regarding the association of p41Plk3 levels with anoikis induction, as shown in our original submission, the increased p41Plk3 expression generated from p72Plk3 cleavage in non-tumorigenic HPDE pancreatic epithelial cells coincided with an increase in the level of apoptosis of cells in suspension culture (Fig. 2b and 2i), suggesting increased p41Plk3 expression is associated with anoikis induction.Direct tests on different PDA lines with different levels of endogenous PLK3, p41Plk3 induction and apoptosis/anoikis were also conducted in supplementary Fig. 10e and 10f.Here, we grow multiple PDAC cells in attached and suspension culture condition to induce anoikis with HPDE included as a control, finding that expression of p41Plk3 was increased in the detached PATC50, 66 and 108 cells compared with attached cells, but remain same in Panc-28, MIA-PaCa-2 and PATC43 cells.(supplementary Fig. 10e).It is worth noting that the response to apoptosis in these cells coincidence with the observed alterations of Plk3 expression.Panc-28, MiaPaCa-2, PATC43 cells had a similar response and resistant to anoikis-induced apoptosis.However, PATC50, 66 and 108 cells responded to detached growth condition and anoikis was induced (supplementary Fig. 10f).Notably, HPDE cells harboring most pronounced p41Plk3 expression under suspension culture exhibited highest apoptosis induction (~50%) compared with PDAC cells (≤40%).These data suggest that the upregulation of p41Plk3 in detached PDAC cells are critical in driving cells towards apoptosis.More susceptible to anoikis in HPDE cell largely depends on its sensitivity to cleavage enabling higher p41Plk3 generation.Therefore, these data also support that induction of p41Plk3 correlates with the level of apoptosis.Supplementary Figure 10.NRDC proteolytic cleavage generates p41Plk3 with pro-apoptotic activity and p38Plk1 with pro-survival function, respectively.(e, f) Immunoblot of p41Plk3 and p68Plk1 (e), and flow cytometry analysis of apoptosis-inducing activity (f) in HPDE and a panel of PDAC cells grown on adhesive (A) or polyHEMA-coated (S) plates.
Considering PDA tumor cells were more resistant to cleavage and anoikis induced by PLK3 expression, in the revision study, we overexpressed Plk3 WT and Plk3R354G containing mutated NRDC cleavage site in several PDAC cells under suspension conditions for detection of anoikis (Fig. 3g, h).The results demonstrated that the generation of p41Plk3 by NRDC cleavage was clearly inhibited to a greater degree in cells transfected with Plk3R354G than in cells transfected with Plk3WT.Moreover, NRDC cleavage site mutation in Plk3 triggered less anoikis as indicated by both reduced PARP cleavage and apoptosis.This suggests that p41Plk3 generation promotes anoikis and its level is associated with anoikis induction level.

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The conclusions for the role of Plk3 in anoikis induction and the mechanism by which Plk3 is proteolytically processed rely heavily on data generated using non-pancreatic cell lines.This weakens the main connection that the authors attempt to make between pancreatic tissues and endogenous Plk3." As discussed in question 1, p72Plk3/p41Plk3-induced apoptosis/anoikis experiments were done in non-tumorigenic HPDE pancreatic epithelial cells in Fig. 2b and 2i, showing induction of p41Plk3 expression led to increased anoikis of cells in suspension culture.The results from supplementary Fig. 10e and 10f with HPDE and multiple PDAC cells grown in attached and suspension culture condition also support that induction of p41Plk3 in PDAC cells with different levels of endogenous Plk3 correlates with the level of apoptosis.In the revision study, we demonstrated that NRDC cleavage site mutation in Plk3 (Plk3R354G) significantly reduced the level of cleaved PARP and apoptosis (Fig. 3g, h).This further confirmed that NRDC-dependent cleavage of Plk3 induces anoikis of PDAC.
3. "Similarly, many of the IP experiments utilize exogenous protein without any attempt to assess proteinprotein interactions using endogenous proteins.This is not to say that there is no role for the IP data with exogenously expressed protein.Rather, the conclusions are overly reliant on these approaches." We thank the reviewer for pointing this out.In the revised manuscript, we performed several IP experiments in PDAC cells using endogenous proteins.4. "Appropriate controls are frequently lacking to confirm overexpression, activity of various inhibitors, and immunoprecipitation.For example, the expression of p110ɑ in Fig. 4i is not confirmed by an immunoblot, and there is no positive control for LY294002 inhibitor activity in Fig. 4. IP experiments often do not include controls for non-specific binding." Thanks for the reviewer to point this out.In the revised study, we confirmed p110a expression in new Fig.4g, added positive controls for LY294002 inhibitor activity in Fig. 4e and 4f, included IgG controls in IP experiments.All experiments are performed in PDAC cells.1b, some of the cell lines labeled on the immunoblot are not mentioned or discussed in the manuscript.This creates difficulty in assessing the conclusions regarding Plk3 levels and the level of aggressiveness of each PDX model."

"In Figure
Plk3 expression in star labelled PDAC PDX cells in Fig. 1b was firstly discussed on page 4 in the manuscript "Strikingly, MDA-PATC148, 148LM, 148LM2, 153, 153LM, and 219B cells, which were derived from liver metastasis, exhibited significantly reduced Plk3 expression compared to nonmetastatic MDA-PATC102, 108, and 124 cells (Fig. 1b), indicating that Plk3 downregulation is associated with PDAC PDX metastasis".Figure legend also explained that "* indicates cells derived from liver metastases of primary PDAC".In Fig 1b, we group these PDX PDAC cell lines into metastatic and nonmetastatic cells.We used metastatic PDX cell line PATC148 and PATC153 in cell models and orthotopic mouse models to demonstrate that activated p41Plk3 induced anoikis and suppressed PDAC tumorigenesis and metastasis (Fig. 7, Supplementary Fig. 9 and Supplementary Fig. 10).In the revised study, we pointed out PATC148 and PATC153 as metastatic PDX cells in the text to strengthen the role of p41Plk3 in inducing anoikis and suppressing PDAC metastasis.
We obtained these 16 PDAC PDX cells from our collaborator Dr. Jason B. Fleming, four of them was characterized in the paper (Kang et al.Lab Invest.2015 Feb; 95(2): 207-222.).We feel it is challenging to conclude Plk3 expression levels and the level of aggressiveness of each SINGLE PDX model based on the western blot in Fig. 1b.6. "CBS loading control band patterns appear substantially different between immunoblots using the same cell lines.For example, compare CBS banding in Fig. 2d & 2e which are both from HPDE cells."Fig. 2d and 2e experiments were conducted in different gels.It should be reasonable to show CBS loading control bands at different molecular weight position for each experiment.This may cause band pattern different between blots.To more rigorously present the data, we repeated the experiment using vinculin or b-actin as loading controls in the revised manuscript (Fig. 2d, e).  7. "The FACS quantifications of apoptotic cells in Fig. 3f-g do not seem to match with the results of the colony formation assay with the same cell lines in Fig. 3d.If there are changes in cell death that are only 10-20%, why is it that colony growth is changing by (in some cases) 90%?" We thank the reviewer for pointing this out.We use two different experiment methods, flow cytometry and colony formation, to measure cell death.The results largely depend on cell starting concentration/seeding density and culture time, which will result in absolute number difference in two different experiments.Importantly, both experiments consistently suggest that NRDC indeed cleaved Plk3 at residue Arg354, p41Plk3 induced high apoptosis comparable to p72Plk3 in 293T cells.The role of activated p41Plk3 in the induction of anoikis was also verified throughout the manuscript using multiple PDAC cells lines and orthotopic mouse models.In the revised study, we also determined the apoptosisinducing ability of Plk3 WT and mutants using colony formation assay and flow cytometry analysis in PANC-1 cells.As shown in Fig. 3c-e, Plk3 especially p41Plk3 promotes anoikis, whereas those with Plk3 DR1 deletion and substitution were anoikis resistant.This suggested that p41Plk3 plays a critical role in inducing anoikis.8. "In Figure 4b, the delta 326-377 protein expression (seen in the input) is substantially lower than the WT or R354G.Thus, the conclusion that the delta 326-377 protein binding to NRDC is compromised is not well supported.It could be that the differences in total protein account for the IP results." We thank the reviewer to point this out.We repeated the IP experiment in PANC-1 cells with even input loading.As shown in figure 4a, deletion of aa326-377 in Plk3 abolished its interaction with NRDC, suggesting that aa326-377 are important for binding to NRDC. 9. "The organization of figure panels throughout the manuscript is muddled and creates difficulty in assessing the findings.Similarly, the figures are not always referenced in the text in order which also creates challenges for readers/reviewers." We have more data included in the revised manuscript.We paid a very close attention to figure organization and presentation.10. "Explanation of intent, description of findings, and interpretation of results are often unclear.It should be easily understood why each experiment was performed, what was found, and how the data support or contradict the hypothesis." In the revised study, we paid more attention to the study design, interpretation of experiment results and other comments as the reviewer suggested.
Minor concerns 1. "Frequently used abbreviations (like PanIN, ORF, PBD) are often not described throughout the paper." In the revised study, we added pancreatic intraepithelial neoplasia for PanIN, and open reading frame for ORF.Polo-box domain for PBD and described them in the text.
2. "The introduction section could contain more information about nardilysin, as it becomes a focal point of the data after not being introduced early in the text." We thank the reviewer for the comment.We do not want to break the flow of our introduction section by inserting NRDC information and there is no developed connection to NRDC early in the text yet.Instead, we introduced more NRDC background in the Results part when we first demonstrated that Plk3 is proteolytically processed by NRDC.
3. "Throughout the paper, Plk1 data are intermingled in figures without much context or justification, especially when the main conclusions relate to Plk3." Five Plks (Plk1-5) have been identified with a conserved protein kinase domain.The role of Plk1 in regulating cell cycle progression and promoting tumor development has been well documented.In this manuscript, our focus is cleavage-mediated Plk3 activation mechanism and p41Plk3 function in suppressing PDAC tumor growth and metastasis.This mechanism plays a similar role for Plk1.Plk1 undergoes the same scission by NRDC proteolytic cleavage to generate p38Plk1, which plays an oncogenic role in PDAC development.We also studied inter-regulation between Plk1 and Plk3 in pancreatic cancer to examine the concurrent opposite effects of Plk3 and Plk1 in regulation of apoptosis.We put the mechanism and functional data of Plk1 side by side with Plk3 to support the discovery of a previously unidentified cleaved form of Plk3 and to suggest the strategy of designing small molecule Plk inhibitors as well.This is further justified in the revised manuscript.We removed the bar graph and keep the table to describe the mice phenotype and statistical analysis in the revised manuscript.5a is difficult to spot at first glance; the scissors blend in too much with the ribbon model."

"The cleavage site in Figure
We modified Figure 5a and made the cleavage site and scissor visible in the revised manuscript (Fig. 5a).
Reviewer #2 -PDAC, metastasis, mouse models -(Remarks to the Author): Title: Nardilysin-Regulated Scission Mechanism Activates Polo-like Kinase 3 to Suppress the Development of Pancreatic Cancer Manuscript Number # NCOMMS-22-33695 "Reviewer's Comments: The focus of this research study indicates the role of Plk3 and its cleaved form of p41KDa mediated anoikis.When p72Plk3 is cleaved by NRDC and p32 Plk3 is removed by degradation, the N-terminal p41Plk3 gains kinase activity.p41Plk3 biological functions include induction of apoptosis and exhibit tumor suppressor role.The findings also demonstrate that PI3K regulates post-translational processing of Plk1 and Plk3 involving PI3K/centaurin-1/NRDC signaling axis to generate functional p38Plk1 and p41Plk3.There are some major concerns about the data presented:" 1. "The focus is on pancreatic cancer, but many results were obtained from 293T human embryonic kidney cell line (Figure 3, 4, 5, 6 and Supplementary Figure 2, 4) and human colorectal carcinoma cell line HCT116 (Supplementary Figure 2, 4)." We thank the reviewer for the comments.The initial study of Plk3 induced anoikis, NRDC-mediated cleavage, identification of cleavage site was carried out using 293T cells, which led to the discovery of p41Plk3.As we did find Plk3 in 293T cells has highest basal level of p41Plk3 generation.These major findings were verified in non-tumorigenic HPDE pancreatic epithelial cells and multiple PDAC cells.Particularly, the role of p41Plk3 induced anoikis was extensively studied in PATC148 and PATC153 cells and their orthotopic mouse models (Fig. 2a-2e, 2i, 3i-3k, 4b-4f, 4g, 4h, 6b-6e, 6k-6n, 7a-7f; supplementary Fig 2g, 6a-6b, 6d-6i, 7d-7f, 8a-8j, 9a-9l, 10a-10h).In the revised study, we performed all experiments in Fig. 3, 4, 5, 6 and Supplementary Fig. 2, 4 using PDAC cells, removed HCT116 results with PDAC cells (Fig. 3f, Supplementary Fig. 2c) and re-organize figures to strengthen our finding that NRDC-mediated cleavage of Plk3 induces anoikis and suppresses pancreatic cancer development.1b-1d: what are the levels of p41Plk3 in the metastatic cell lines?Supplementary figure 1c: whether -actin is analyzed on the same membrane?What are the levels for p41Plk3?"

"Figure
In the revised study, we grow multiple PDAC cells in suspension culture condition.As shown in Fig. 2h, metastatic PDX cells exhibited lower level of p72Plk3 expression and undetectable p41Plk3 compared to HPDE and non-metastatic PDX cells, indicating that Plk3 in metastatic PDX cells are more resistant to cleavage and anoikis induction.Downregulation of p72Plk3 and p41Plk3 is associated with PDAC PDX metastasis.We used antibody specific for N-termini of Plk3 (anti-human) in this experiment.
For Fig. 1d, we have attempted to examine p41Plk3 expression in PDAC cells isolated from p48cre;Kras LSL-G12D ;INK4a/Arf F/F mice (KIC) and KrasLSL-G12D; p53LSL-H172R mice (KPC) and in normal mouse pancreatic epithelial cells (MPECs) using anti-mouse, N-terminal specific Plk3 antibody.However, due to the poor antibody quality, the blot showed many non-specific bands and did not give any informative result.This is not presented in the manuscript.Supplementary Fig 1c, -actin is analyzed on the same membrane.Fig. 1b showed that MDA-PATC148, 148LM, 148LM2, 153, 153LM, and 219B cells, which were derived from liver metastasis, exhibited significantly reduced Plk3 expression compared to non-metastatic MDA-PATC102, 108, and 124 cells, indicating that Plk3 downregulation is associated with PDAC PDX metastasis (page 4)".As addressed in the question 1 above, our new Fig.2h showed metastatic PDX cells exhibited lower level of p72Plk3 expression and undetectable p41Plk3 compared to HPDE and nonmetastatic PDX cells, supporting the notion that downregulation of p72Plk3 and p41Plk3 is associated with PDAC PDX metastasis.This is also consistent with our genetic evidence that deleting Plk3 in p48cre;Kras LSL-G12D mice dramatically accelerated Kras G12D -driven PanIN and subsequent PDAC formation.Metastases in the liver and lung were observed in 7 of the 11 (64%) mice that developed PDAC (Fig. 1f-1h, supplementary Fig. 1d and 1e).These results suggest that loss of Plk3 is involved in PDAC tumor progression and metastasis.
We have demonstrated in the manuscript that p41Plk3 expression promotes cell detachmentinduced apoptosis, a typical feature of anoikis.Anoikis is a programmed cell death occurring upon cell detachment from the correct extracellular matrix, thus disrupting integrin ligation.It is a critical mechanism in preventing dysplastic cell growth or attachment to an inappropriate matrix.Anoikis prevents detached epithelial cells from colonizing elsewhere and is thus essential for tissue homeostasis and development.Cancer cells acquire anoikis resistance to survive after detachment from the primary sites and travel through the circulatory systems to spread throughout the body.We stressed on anoikis and aim at the molecular mechanisms governing p41Plk3-induced anoikis and focusing on their regulation in metastatic PDAC.Plk3 in PDAC cells are more resistant to NRDC-mediated cleavage to generate activated p41Plk3, bestowing PDAC cells to develop anoikis resistance, to progress towards malignancy and metastasis.4. "What is the status of p41Plk3 in the KPC mice in Figure 1d? Figure 1h: is there an IHC staining done to show the expression of p41Plk3 in p48-cre; KrasLSL-G12DPlk3+/+." Please refer to our answers to Q3 above for p41Plk3 level in Fig 1d .It is a great idea to identify p41Plk3 by IHC in tissues, as it will provide direct evidence that Plk3 is in fact cleaved in human or mouse tissues.However, this assay is obstructed by the availability of Plk3 antibody that can specifically recognize p41Plk3 in IHC considering both p41Plk3 and p72Plk3 are recognized by N-terminus Plk3 antibodies.The current available Plk3 commercial antibodies would not distinguish p41Plk3 from p72Plk3 in IHC.Instead, we used Patient-derived xenograft (PDX) models throughout our research, which were obtained from our collaborator Dr. Jason Fleming at the Department of Surgical Oncology at MD Anderson Cancer Center.PDX cells derive directly from pancreatic cancer patient tumors tissues.That reflects original tumor characteristics such as heterogeneous histology, clinical biomolecular signature, malignant phenotypes and genotypes, tumor architecture, and tumor vasculature, making them a valuable foundation of the oncology research.We grow multiple PDX cells under suspension culture and revealed expression of p41Plk3 under physiological conditions, validating the cleavage of Plk3 is the mechanism for generating p41Plk3 (Fig. 2h, 4j and supplementary Fig 9e).
It would be of great interest to generate C-terminus specific antibodies to detect C-terminal carboxyl group of cleaved p41Plk3 in the future study.But we also realized it takes too long time to make such antibodies for manuscript revision.As monoclonal antibodies against the unique charged C-terminal carboxyl group are required, the screening process will be very time consuming.Furthermore, it is unclear whether posttranslational modification of C-terminus (C-methyl-esterification and α-amidation) will hinder the binding of C-terminus specific antibodies and complicates interpretation of the expected results.For examples: C-terminal α-amidation neutralizes the negative charge of the carboxyl group at the C-terminus.C-methyl-esterification is the most frequently annotated C-terminal modification as reported in TopFIN.

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In figure 1, most of the pancreatic cell lines still express a detectable p72Plk3 level.How do you justify this?Whether it determines the possibility of Plk3 independent tumor mechanism in PDAC or if there is any possible mechanism involved in inhibiting the function of p72Plk3.What's the expression pattern of NRDC in pancreatic cancer?"These are excellent questions.Western blot in Fig. 1b and 1c showed that Plk3 protein levels were reduced in most human PDA PDX cells, particularly in PDXs derived from liver metastases, and were also decreased in a subset of human PDA cell lines compared to normal HPDE cells.The mechanism investigations showed that NRDC/PI3K-regulated cleavage of Plk3 generates a kinase-active p41Plk3 fragment that can induce anoikis in pancreatic cancer cells, reduce growth and metastasis in mice.We found that PDA tumor cells, especially metastatic PDX PDAC cells were more resistant to cleavage and anoikis induced by PLK3 expression.To demonstrate whether the cleavage of Plk3 is associated with NRDC or PIP3 expression levels, we performed western blot analysis using a panel of PDAC cell lines under suspension culture (Fig. 4h).The results showed that the cleavage of p72Plk3 positively correlates with expression level of NRDC.Detached HPDE cells exhibited high level of p110 compared with PDAC cells.For reviewer information, our bioinformatics analysis using ICGC datasets showed the expression of Plk3 and NRDC is associated (r=0.311,p=0.002) (see below).

Expression correlation between Plk3 and NRDC in PDAC samples
Linking a study from Ikuta et al that [1] pancreatic deletion of NRDC in Kras G12D -driven mice promoted PDAC tumorigenesis and NRDC expression is absent in a subset of human PDAC, our studies suggest a fundamental mechanism that, in addition to reduced expression level, deficient NRDC-mediated cleavage on Plk3 to generate activated p41Plk3 might bestow PDAC cells to develop anoikis resistance, to progress towards malignancy and metastasis.2b cleaved PARP induction is visibly seen after 2 hrs in suspension culture.Whereas figure 2i, 2j, 2k -HPDE/HEK293T/MEF cell lines reveal the cleavage to 41kd from 72 takes at least 3 hrs.Whether the induction of cleaved PARP is independent of 41kd?"

"In figure
We thank the review for the comments.However, we would not make this conclusion based on band intensity as these western blots are performed using different cell lines, and they target different proteins (cleaved PARP vs p41Plk3).We have shown that the expression of Plk3 in HEK293T cells led to cell round-up, detaching from plate, floating and apoptosis (anoikis) (Supplementary Fig. 2a and 2b).The floating cells exhibited higher level of cleaved PARP than attached cells.While the vector-transfected 293 cells grown under attached and suspension condition do not exhibit cell death (Supplementary Fig. 2a  and 2b).Further investigation of the mechanism through which PLK3 induces apoptosis led to the discovery p41Plk3 with the level of p41PLK3 being higher in floating cells undergoing apoptosis (Supplementary Fig. 2b and 2i), these data suggest that the occurrence of p41Plk3 causes cell detachment-induced apoptosis, anoikis.
In the revised manuscript, to validate the essential role of p41Plk3 in inducing anoikis, we determined the effect of Plk3 deletion on PDAC cell anoikis (Fig. 2l, m).The result demonstrated that knockout of Plk3 expression in several PDAC cells led to decreased expression of p41Plk3 and subsequent reduced level of cleaved PARP and apoptosis-inducing activity (Fig. 2l, m).Regarding the association of p41Plk3 levels with anoikis induction, as shown in our original submission, the increased p41Plk3 expression generated from p72Plk3 cleavage in non-tumorigenic HPDE pancreatic epithelial cells coincided with an increase in the level of apoptosis of cells in suspension culture (Fig. 2b and 2i), suggesting increased p41Plk3 expression is associated with anoikis induction.Additionally, we overexpressed Plk3 WT and Plk3R354G containing mutated NRDC cleavage site in several PDAC cells under suspension conditions for detection of anoikis (Fig. 3g, h) in the revised study.The results demonstrated that the generation of p41Plk3 by NRDC cleavage was clearly inhibited to a greater degree in cells transfected with Plk3R354G than in cells transfected with Plk3WT.Moreover, NRDC cleavage site mutation in Plk3 triggered less anoikis as indicated by both reduced PARP cleavage and apoptosis.This suggests that p41Plk3 generation promotes anoikis and its level is associated with anoikis induction level (cleaved PARP level).

Fig. 3 Identification of the proteolytic cleavage site in the DR1 domain of Plk3. g, h Immunoblots of p72Plk3 and p41Plk3 expression (g) and flow cytometry analysis of apoptosis-inducing activity (h) from
PDAC cells transfected with indicated Flag-tagged Plk3 WT or mutants and grown under suspension condition for 36 h.

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The mouse developed a tumor in p48cre; KrasLSL-G12DPlk3+/+ group, did it show any correlation with the genes mentioned in supplementary Table 1? Did authors analyze those samples because they should match with the genes mentioned in the surviving colonies that are resistant to Plk3 overexpression?" We thank reviewer for raising these interesting questions.ORFs isolated from the 6 out of 60 surviving colonies in supplementary Table 1 share common features, encoding metalloendopeptidase nardilysin (NRDC) cleavage sequences -X-Arg-Lys-motif.Functionally, they might interfere with nardilysin-mediated cleavage of Plk3.If so, these peptides are great research tools, as they might promote pancreatic cancer development.However, we feel it would be beyond the scope of this manuscript revision to study these 6 genes, especially this manuscript is centered on our novel finding that Plk3 is activated by PI3K/NRDCregulated cleavage and p41Plk3 promote anoikis, G2/M arrest, and PDAC and metastasis suppression.8. "What causes Plk3 downregulation?Is there any known mutation?And what percentage of PDACs in which Plk3 is downregulated?If Plk1 seems to be an oncogene, is there an interplay between the two family members?"These are very interesting questions.Please refer to question 5. Our studies suggest a fundamental mechanism that, in addition to reduced Plk3 expression level, deficient NRDC-mediated cleavage on Plk3 may cause reduced induction of activated p41Plk3, leading to PDAC cells anoikis resistance.In regard to mutation, to our knowledge, there is no evidence for tumor-associated inactivating mutations in Plk3 or NRDC in pancreas or other cancers reported in the literatures or database.
Additionally, Kras G12D is the earliest mutation detected in pancreatic intraepithelial neoplasia (PanIN) lesions and found in about 90% of human PDAC.We have planned to study whether Kras mutation affects Plk3 expression and cleavage in the revised study.We generated construct with Doxycycline-inducible Kras expression in HPNE cells and analyzed whether Kras regulates Plk3 expression; whether cFos is involved in Kras-Plk3 pathway.For reviewer information, Panel A showed that doxycycline induced expression of Kras stimulates an increased expression of Plk3 in first 24h, followed by a decreased expression over the next 48 and 72h.c-Fos showed delayed expression reduction in response to Kras induction.We also analyzed c-Fos mRNA levels in multiple PDAC cells carrying Kras mutation by qRT-PCR and the results showed c-Fos expression was significantly lower in 7 out of 12 of these PDAC cells than in HPDE cells (Panel B).This indicates Kras might regulate Plk3 expression and suppression of PLK3 expression in these cells is a result of reduced c-Fos levels.In addition, we have previously shown that Plk3 expression was regulated by RelA/NF-B [8].Reduced RelA/NF-B and c-Fos levels could result in the decreased Plk3 expression.This deserves future detailed study.Regarding the percentage of downregulated Plk3 in PDACs, we have performed immunohistochemistry staining for Plk3 expression in human PDACs using a tissue microarray containing 33 PDAC samples and 56 normal pancreatic tissue samples (Fig. 1a).The results showed that 45% (25/56) of the normal tissue samples exhibited high levels of Plk3 expression, 82% (27/33) of the PDACs had low expression of Plk3 (p = 0.0114).
It is interesting to study molecular interplay between Plk1 and Plk3 that regulate opposing effects in PDAC development.Our preliminary data suggested that p41Plk3 induced anoikis by increasing expression of pro-apoptotic genes.Conversely, highly expressed Plk1 in PDAC tumor promoted antiapoptotic response [2][3][4].Since both the activations of Plk1 and Plk3 are regulated by NRDC cleavage, we explored whether there is inter-regulation between Plk1 and Plk3 by stably expressing both Plk1 and Plk3 together in PATC148 cells.As shown in our original submission (supplementary Fig. 10g and 10h), The increased p41Plk3 expression may suppress the expression of p68Plk1, resulting in decreased levels of p38Plk1 compared to p68Plk1 over-expression alone (supplementary Fig. 10g).This consequently led to enhanced anoikis activity in co-transfected cells (supplementary Fig. 10g and 10h), implying a mutually exclusive inter-regulation of Plk1 and Plk3 proteolytic cleavage in controlling PDAC cell proliferation and apoptosis.Supplementary Figure 10.NRDC proteolytic cleavage generates p41Plk3 with pro-apoptotic activity and p38Plk1 with pro-survival function, respectively.(e, f) Immunoblot of p41Plk3 and p68Plk1 (e), and flow cytometry analysis of apoptosis-inducing activity (f) in HPDE and a panel of PDAC cells grown on adhesive (A) or polyHEMA-coated (S) plates.9. "Co-IP of NRDC indicated that LY294002 increased the binding of NRDC to centaurin-1 in the cytoplasm and decreased its binding to p72Plk3 (Fig. 4i).But it was shown only in 293T, which is a human embryonic kidney cell line, and not in any other PDAC cell line.Though the experiments with LY294002 were performed in many different cell lines, the Western for centaurin-1 was shown only in 293T cells." We repeat this IP experiment using PANC-1 cell line in the revised study.As shown in the Fig. 4j, Co-IP of NRDC indicated that LY294002 increased the binding of NRDC to centaurin-1 in the cytoplasm and decreased its binding to p72Plk3.In the revised study, we determined half-life of Plk3CT in metastatic PATC148 cells (Fig. 5i, j).Time course of cycloheximide (CHX) treatment in PATC148 cells expressing NT1-353-and CT 354-646 -Plk3 revealed that NT1-353 was stable over the time, whereas CT354-646 is subjected to degradation with half-life of 12-13 h (Fig. 5i, j).Plk3NT1-353 is tagged with Flag and detected in western blot using anti-Flag; Plk3CT354-646 is detected using C-terminal specific anti-Plk3 antibody.
1. "Supplementary Figure 7h -please mark the different cell cycle phases and include population distributions." We marked cell cycle phase in Supplementary Fig. 8h in the revised manuscript.Cell cycle distribution was studied using flow cytometry and results were shown in Supplementary Fig. 8a, revealing overexpression of p41Plk3 induced G2-M arrest, whereas p72Plk3 had no effect on cell cycle distribution.
2. "Authors have found that p41Plk3 expression is significantly increased in the PTEN-KO compared to PTEN-WT MEF cells.It is also suggested that PI3K is required to cleave p72Plk3 into its active form p41Plk3 (apoptosis inducer).-If p72Plk3 is a tumor suppressor and its active form p41Plk3 is anoikis or apoptosis inducer, then the concern is about PI3K because PI3K oncogenic signaling is known to be upregulated in PDAC.PTEN which is a known tumor suppressor KO increased p41Plk3 expression.Please justify how to correlate these events." We thank the reviewer for pointing this out.We also realized that PI3K oncogenic signaling is known to be upregulated and PTEN can downregulate PI3K in PDAC.LY294002 treatment indeed induced apoptosis as reflected by increased PARP cleavage, but also blocked Plk3 cleavage (Fig. 4e-g).
Firstly, considering the activated PI3K/NRDC in PDAC also regulates p68Plk1 cleavage to generate p38PLK1, which has anti-apoptotic activity (supplementary Fig. 10a-10d).The low induction and minimum change of p41Plk3 expression regulated by PI3K may not be sufficient to counteract p38Plk1 pro-proliferation effects in PDAC.
Secondly, considering oncogenic Kras activates PI3K and other signaling pathways driving pancreatic tumorigenesis, our results suggest Plk3 cleavage may not be required for the effects of PI3K in PDAC development.There could be other molecules involved in the regulation of Plk3 cleavage.
Thirdly, additional examples can be appointed out: It is clear that NF-KB is anti-apoptotic or proapoptotic bifunctional signaling factor, depending on upstream stimulation.c-Fos is generally thought to be oncogenic but could also have pro-apoptotic function as shown in our study.Our study began to suggest that phosphorylation on Thr-164 of c-Fos by Plk3 and upregulation of pro-apoptotic downstream target genes contributed the functional switching.As our results showed, PI3K may have a potential to be a bifunctional signaling regulator.However, the molecular mechanisms that control this functional switching is not known.Respectively, we would like to point out that exploration of the novel regulation mechanisms of PI3K is out of scope of our current study.10. "Plk1 cleavage is markedly increased in PTEN-KO but not in PTEN-WT MEFs.If Plk1 is a tumor promotor.It's been known that PTEN is a negative regulator of PI3K and PI3K is a known downstream effector of K-Ras, which is a driver of PDAC.Then how the KO of PTEN increases the cleavage of Plk3 into the active pro-apoptotic p41Plk3 KDa form?And vice versa how the inhibition of PI3K by LY294002 prevents the cleavage of p41plk3, which can induce apoptosis?"Please refer to the question 12 above.11. "Then if Plk1 is a tumor promotor and Plk3 is a tumor suppressor?What is the NRDC binding affinity to each." Our results suggested that NRDC interacts with Plk1 and Plk3 and generated NRDC-cleaved p38Plk1 and p41Plk3 kinases.In vitro experiments mixing purified Plk with NRDC and determining which Plks will bind to NRDC more is one of the approaches to understand which Plk to be cleaved by NRDC.However, using purified proteins in large excess (for visualization purpose) is not physiological condition and the potential mechanisms derived from those in vitro study are not accurate.In our preliminary study to determine binding affinity between NRDC and Plk1/Plk3 using Microscale thermophoresis (MST) method, 1/3-1/2 Plk3 protein has already been cleaved to generate p41Plk3 (aa1-353) during the expression and purification of Plk3.Our results also showed aa326-377 of Plk3 is important for interaction with NRDC (Fig. 4a).Therefore, a mixture of p41Plk3 (lost important fragment to bind NRDC) and p72Plk3 might not be stringent for the binding affinity measurement.It may be a better approach to use differential tagged Plk1, PLk3, and NRDC isolated from knock-in mouse model as a starting point for biochemical analysis.
Reviewer #3 -Plks, mass-spec -(Remarks to the Author): "The manuscript was from the labs of Drs.Paul Chiao, Peter Stambrook and El Mustapha Bahassi, important contributors of the polo kinase field for over many years.The authors clearly deserved to be congratulated as this is another nice and important piece of contribution to the field.
The authors aimed to dissect the regulation of Plk3 in pancreatic cancer in great detail.Indeed, the regulation of Plk3 remained in the dark since its discovery and complete cloning in 2000 (Oncogene.2000 Oct 5;19(42):4832-9.doi: 10.1038/sj.onc.1203845).At first the authors analyzed the expression pattern of Plk3 in clinical samples and in the transgenic mouse models P48-Cre; KrasLSL-G12D; PLK3+/+ vs. Cre; KrasLSL-G12D; PLK3-/-.Upon overexpression of 72-kDa Plk3 associated with the loss of anchoragedependent growth (high percentage of floating cells) a subfragment of Plk3 (p41Plk3) encompassing the kinase domain became visible.Plk3-induced anoikis correlated with increasing levels of p41Plk3.Most interestingly, the authors could identify via functional screening of an ORFs library the metalloendopeptidase nardilysin (NRDC) that is responsible for the cleavage of Plk3 into two subfragments.It is also of broad relevance that this mechanism plays a similar role for Plk1 cleavage.Furthermore, the regulation of NRDC by PI3-kinase was investigated in detail.C-Fos could be identified as novel substrate for Plk3 in its p41Plk3 form.
In summary, the data are of very high relevance for the development of pancreatic cancer and for the entire field of polo kinases.In addition, for the use of small molecule Plk inhibitors in clinical trials in relation to their specificity the new data by Fu et al. need to be considered." A few aspects should be addressed before publication 1. "Is there a correlation between mRNA levels of Plk3 and the corresponding protein in human pancreatic cancer (Fig. 1, Suppl.Fig. 1)?The authors should clearly state in which type of cancer cells (primary tissue, PDX cells etc.) this correlation was seen and what technique and which antibody was used.This aspect is important because there is a long-lasting debate about the quality of Plk3 antibodies."Supplementary Fig. 1b, in our earlier studies, Plk3 mRNA is extracted from primary human PDAC and adjacent normal pancreatic tissues; Fig. 1b and supplementary Fig. 1c, Plk3 protein is from newly established PDAC PDX cells.They are not correlated.For reviewer information, we performed q-PCR to determine mRNA levels of Plk3 in PDAC PDX cells.The results demonstrated that in comparison to nontumorigenic human pancreatic duct epithelial (HPDE) cells, Plk3 expression was remarkably decreased in PDAC cells at mRNA level.However, it does not show a correlation between the varied mRNA levels of Plk3 and the corresponding protein among PDAC cells.
As the reviewer pointed out, specificity of PLK3 antibodies is an issue, especially for antibodies specific for N-termini of Plk3.Part of Plk3 antibodies (BL 1696-1699) characterized in supplementary Fig. 2f were discontinued during our study.We used specific siRNAs targeting PLK3 to monitor the endogenous expression of full-length PLK3 and p41PLK3 in PDAC cells with antibodies specific for N-or C-termini of Plk3 (Supplementary Fig. 2g).These antibodies were used in subsequent analyses.In the Figures of the revised manuscript, we clarified Plk3 antibodies used for different experiments.
2. "Indeed, a role for Plk3 in cell cycle regulation has been discussed multiple times but not for the G2/M transition or the duration of mitosis.Thus, it is surprising that an overexpression of p41Plk3 induces a high percentage of cells (over 80%) that are stuck in mitosis.I think it would be beyond the scope of the manuscript to investigate the mechanism that is responsible for this mitotic arrest, but at least potential mechanisms should be discussed." The occurrence of aberrant mitosis during p41Plk3-induced high percentage of cell death suggest cells might undergo mitotic catastrophe (MC), a type of cell death occurring during mitosis as a result of DNA damage.During MC, tetraploid cells with a range of different nuclear morphologies from binucleated to multi-micronucleated formed.Mitotic catastrophe has long been considered as a mode of cell death that results from premature or inappropriate entry of cells into mitosis and can be caused by chemical or physical stresses.Mitotic catastrophe is one way in which cells prevent the propagation of genome unstable cells.If mitotic catastrophe fails for cells whose genome has become unstable they can propagate uncontrollably and potentially become tumorigenic.The involvement of p41Plk3 in the mitotic catastrophe might play an important role to prevent the proliferation of cancerous cells to develop of tumors.
We discussed the ptential involvement of p41Plk3 in the mitotic catastrophe in the revised manuscript.

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The major topic of this manuscript is the regulation of cell death by Plk3.The exact cell death pathways and mechanisms induced by Plk3 are still a matter for future research.In a previous study (Cell Res.2016 Aug;26(8):914-34.doi: 10.1038/cr.2016.78) the role of Plk3 for the regulation of the extrinsic cell death was explored.Would it be possible that p41Plk3 phosphorylates caspase-8 or other cell death transducing proteins in pancreatic cancer cells?Has this been explored?" We have demonstrated that p53 is another PLK3 substrates that are important for eliciting the apoptotic response [4][5][6].Previously, we showed that Plk3 formed a complex with p53 and was involved in the phosphorylation of p53 on Ser-20 in response to superoxide.Inhibition of Plk3 expression by Plk3 small interfering RNA suppressed the superoxide-mediated apoptosis.Overexpression of wild-type Plk3 in HCT116 p53+/+ cells induced rapid apoptosis, whereas overexpression of wild-type Plk3 in HCT116 p53-/-cells and the kinase-defective mutant Plk3(K91R) in p53+/+ cells induced delayed onset of apoptosis.Furthermore, mutagenesis of Plk3 showed that the N-terminal domain (amino acids 1-26) is essential for the induction of delayed onset of apoptosis.Together, we have demonstrated that Plk3 induces apoptosis through phosphorylation of p53 protein at Ser-20, possibly involving the Src homology 3 domain in the N terminus of Plk3 to initiate proapoptotic signaling [7].It will be interesting to explore p41Plk3 phosphorylation on caspase-8 or other cell death transducing proteins in the future research.
We thank the authors for their responses.While many of the concerns have been sufficiently addressed, there remain a few important questions that were not satisfactorily answered.
1.The conclusions regarding Plk3 and anoikis are still not satisfactorily supported.Supplementary Fig. 2A-2B show that Plk3 overexpression causes a significant increase of cell death in attached cells.While the point that the cells are observed to be detached is noted, the observation that these cells are floating does not support a role for Plk3 in anoikis (defined as caspase-dependent death caused by detachment).These cells could simply be lifting off the plate as they die by apoptosis and the lack of ECM attachment is not a trigger.It is acknowledged that the authors have softened their language regarding proteolytic processing of Plk3 being "essential" for anoikis.However, the new panels in Figures 2l-m dont build much on the original submission as the correlative nature of the p41 fragment with cell death remains an issue in the revised manuscript.Controls for the effect of Plk3 in attached cells are not present in the new panels.As such, the importance of the proteolytic processing of Plk3 on anoikis remains correlative and devoid of causality.
2. The use of MEF and HEK-293T cells, which are non-pancreatic and non-cancerous cell lines, still heavily contribute to important conclusions regarding the cleavage of Plk3 and its effect on apoptotic cell death in pancreatic cancer (Fig. 2f-g, j-k; Supp.Fig. 2b, e, i-j).
3. I remain confused about the large difference in cell death observed in Figure 3 (90% for colony formation, just ~10-20% flow cytometry).The authors point out differences in concentration and seeding density, but dont really address the discrepant nature of these findings or consider possible alternative interpretations for this large difference.For example, perhaps activated p41Plk3 is causing an antiproliferative effect?4. PDX lines 107 and 216 are shown in Fig. 1b, but they are not addressed in the text with other lines referenced in the text and author's response.They were also not characterized in the paper referenced by the authors (Kang et al, 2015).As stated in our original review, these lines are apparently nonmetastatic and have no Plk3.The authors state in their response it is "challenging to conclude Plk3 expression levels and the level of aggressiveness of each single PDX model based on the western blot in Fig. 1b".Fair point, but yet the authors continue to do precisely this.They state: "Furthermore, we examined the expression of Plk3 in 16 newly established PDAC PDX cell lines 30 and demonstrated that Plk3 protein levels were remarkably decreased in PDX cells than in nontumorigenic human pancreatic duct epithelial (HPDE) cells (Fig. 1b and Supplementary Fig. 1c).Strikingly, MDA-PATC148, 148LM, 148LM2, 153, 153LM, and 219B cells, which were derived from liver metastasis, exhibited significantly reduced Plk3 expression compared to non-metastatic MDA-PATC102, 108, and 124 cells (Fig. 1b), indicating that Plk3 downregulation is associated with PDAC PDX metastasis." If the conclusion is that you can't use the loss of Plk3 in PDX 107 and 216 to make conclusions about aggressiveness/metastasis, then how can the authors confidently make the statement above?Reviewer #3 (Remarks to the Author): reviewer #3: ad 2) pages 15/16.The discussion on mitotic catastrophe is very vague.Considering the high percentage of cells in mitosis (>80%) it would be easy to determine whether mitotic catastrophe can be found in the vast majority of mitotic cells induced overexpression of p41Plk3.
ad 3) The authors cite previous papers on the role of PLK3 and p53 in colon cancer cells.Whether Nardilysin-Regulated Scission Mechanism activated PLK3 induces apoptosis via phosphorylation of p53 requires detailed investigations.In addition, a high percentage of p53 (>60-70%) is mutated in pancreatic cancer.Whether the mutated form of p53 in PC is phosphorylated by p41PLK3 remains elusive.At least it should be discussed that other mediators of apoptosis like phosphorylation of Caspase-8 by PLK3 could also be in the observed apoptosis.

Fig. 2
Fig. 2 Proteolytic processing of p72Plk3 to generate p41Plk3 is important for induction of anoikis.l, m Immunoblot of p72Plk3, p41Plk3, and cleaved PARP expression (l) and flow cytometry analysis of apoptosis-inducing activity (m) of indicated PDAC cells that were lentivirally transduced to express the sgRNA targeting Plk3 or non-targeting control sgRNA.

Fig. 2
Fig. 2 Proteolytic processing of p72Plk3 to generate p41Plk3 is important for induction of anoikis.b Immunoblots of cleaved PARP in HPDE cells grown in suspension (Sus.Culture) on polyHEMA-pre-coated plates at the indicated times.I Immunoblots of p72Plk3 and p41Plk3 in HPDE cells grown in suspension for the indicated times.

Fig
Fig. 3 Identification of the proteolytic cleavage site in the DR1 domain of Plk3.g, h Immunoblots of p72Plk3 and p41Plk3 expression (g) and flow cytometry analysis of apoptosis-inducing activity (h) from PDAC cells transfected with indicated Flag-tagged Plk3 WT or mutants and grown under suspension condition for 36 h.

Fig. 4
Fig. 4 Phosphoinositide 3-kinase regulates NRDC activity for Plk3 and Plk1 activation via centaurin-1.e, f Immunoblot of p72Plk3, p41Plk3, and cleaved PARP expression in PATC148/ip72Plk3 cells (e) and p72Plk3 transfected PANC-1 cells (f) treated with PI3K inhibitor LY294002 (20 µM) for indicated times.g p72Plk3 and p41Plk3 expression in PANC-1 cells co-transfected with Plk3 and a vector control or the PI3K catalytic unit p110.j IP and IB detection of increased binding of NRDC with -centaurin and decreased binding of Plk3 with NRDC in PANC-1 cells transfected with Flag-p72Plk3 and treated with or without LY294002 (32 M).

Fig. 3
Fig. 3 Identification of the proteolytic cleavage site in the DR1 domain of Plk3.c-e Colony-formation assay (c, d) and flow cytometry analysis of apoptosisinducing activity (e) from PANC-1 cells transfected with indicated Flag-tagged Plk3 or mutants.

Fig. 2
Fig. 2 Proteolytic processing of p72Plk3 to generate p41Plk3 is important for induction of anoikis.d, e Immunoblots of cleaved PARP in HPDE cells with stable shRNA-mediated knockdown of Plk3 (d) and stably reconstituted with Flag-Plk3 (e).

Fig. 2
Fig. 2 Proteolytic processing of p72Plk3 to generate p41Plk3 is important for induction of anoikis.h Immunoblots of p72Plk3 and p41Plk3 in a panel of PDX cell lines grown in suspension culture for 48 h.

Figure 4 .
Figure 4. (h) Immunoblots of p41Plk3, NRDC and p110 expression in a panel of PDX cell lines in comparison to HPDE cells.Cells were grown in suspension culture for 36 h.

Fig. 2
Fig. 2 Proteolytic processing of p72Plk3 to generate p41Plk3 is important for induction of anoikis.l, m Immunoblot of p72Plk3, p41Plk3, and cleaved PARP expression (l) and flow cytometry analysis of apoptosis-inducing activity (m) of indicated PDAC cells that were lentivirally transduced to express the sgRNA targeting Plk3 or non-targeting control sgRNA.

Fig. 2
Fig. 2 Proteolytic processing of p72Plk3 to generate p41Plk3 is important for induction of anoikis.b Immunoblots of cleaved PARP in HPDE cells grown in suspension (Sus.Culture) on polyHEMA-pre-coated plates at the indicated times.I Immunoblots of p72Plk3 and p41Plk3 in HPDE cells grown in suspension for the indicated times.

(
A) qRT-PCR analysis of Kras, Plk3 and c-Fos expression under a Dox-inducible system with Kras stably transfected HPNE cells treated with Dox for the indicated times.(B) qRT-PCR analysis of c-Fos expression in a panel of PDX cell lines and HPDE cells.

Fig. 4
Fig. 4 Phosphoinositide 3-kinase regulates NRDC activity for Plk3 and Plk1 activation via centaurin-1.j IP and IB detection of increased binding of NRDC with -centaurin and decreased binding of Plk3 with NRDC in PANC-1 cells transfected with Flag-p72Plk3 and treated with or without LY294002 (32 M).
0. "Figure5i: What is the protein half-life of plk3NT/plk3CT in the metastasis cell lines?Whether different antibodies are used for detecting NT-1-354 and CT-356-646?"

Fig. 4
Fig. 4 Phosphoinositide 3-kinase regulates NRDC activity for Plk3 and Plk1 activation via centaurin-1.e, f Immunoblot of p72Plk3, p41Plk3, and cleaved PARP expression in PATC148/ip72Plk3 cells (e) and p72Plk3 transfected PANC-1 cells (f) treated with PI3K inhibitor LY294002 (20 µM) for indicated times.g p72Plk3 and p41Plk3 expression in PANC-1 cells co-transfected with Plk3 and a vector control or the PI3K catalytic unit p110.j IP and IB detection of increased binding of NRDC with -centaurin and decreased binding of Plk3 with NRDC in PANC-1 cells transfected with Flag-p72Plk3 and treated with or without LY294002 (32 M).